Signal-generating techniques are often employed to detect chemical reactions, biological events, and physical and chemical properties of a sample. Typically, the signal is in the form of radiation (e.g., light, color, fluorescence, luminescence, particle emissions) and either is a component/product of the reaction or is generated upon interaction of a component/product with an added indicator moiety.
An example of the use of signal generation to detect a biological substance is the quantitation of an antigen in biological samples using enzyme-linked immunosorbent assays (ELISA). In these assays, a sample is exposed to an enzyme-conjugated antibody capable of binding to the specific antigen to be detected. The conjugated enzyme is one that catalyzes a reaction which generates a signal (e.g., color, fluorescence, luminescence) that can be directly correlated with the amount of antigen in the sample. This type of assay, in which the property to be measured is constant and the signal is sustained, is referred to as an endpoint assay. Thus, in these assays, the signal is allowed to develop over time, and then a single signal measurement is taken after the reaction is complete in order to quantify the property.
In contrast to attributes that can be measured in endpoint assays, there are many properties, reactions and biological events that are dynamic and transient and/or rapidly occurring. For example, many cellular processes are rapid and transient in nature. Cells receive stimuli from the environment and must respond immediately for proper function and survival. Modulation of cell receptors and ion channels by binding of ligands can result in cellular responses such as changes in the levels of intracellular second messengers (Ca2+, cyclic nucleotides, etc.). For instance, activation of a cell surface calcium channel upon binding of a ligand causes the channel to open and results in a rapid inward flux of calcium that transiently increases the intracellular Ca2+ concentration which rapidly declines to pre-activation concentrations. If the cell has been pre-loaded with a Ca2+-sensitive fluorescent indicator, the change in intracellular Ca2+ appears as a rapid increase and then decrease in fluorescence of the cell.
Because signal-generation techniques can provide information regarding the actual functioning of a cell, it is desirable to attempt to apply these methods to the identification of compounds that influence cellular activities (e.g., potential drugs that affect cell function through interaction with cell receptors, ion channels or enzymes). However, in drug screening procedures, large number of compounds are tested for cell modulation before even a small number are identified as potential drugs. The problems faced in using signal-generation techniques to detect and measure such transient and/or rapidly occurring phenomena in a single assay are only compounded in attempting to apply these techniques to the simultaneous performance of multiple assays for rapid screening of thousands of compounds.
For instance, the signal generated in these assays is rapidly occurring and transient, as is the phenomenon itself. Thus, in these assays, if initiation of the reaction or event (e.g., activation of the calcium channels by addition of ligand) is not coordinated with almost immediate signal detection in a dynamic fashion, the signal may reach a maximum and diminish before it is detected. In order to perform large-scale compound screening, coordination of sample handling and signal detection must be accomplished for many assays simultaneously. Furthermore, it is desirable to obtain a real-time record of each event until it has progressed to a point beyond that of maximum signal change. Thus, the duration, as well as the timing, of signal measurement poses an additional complication in these assays since the signal must be measured essentially constantly.
Accuracy of signal measurement is particularly critical in high-throughput screening assays of thousands of compounds. The need to perform a multitude of individual compound tests in a limited amount of time prohibits replicate assays of each compound. Additionally, sensitivity of signal detection presents another difficulty in signal-based assays of these phenomena. The signal changes accompanying these reactions or events are not only transient changes in the relative levels of the signal (i.e., increases in signal above a baseline level of signal), but may also be of relatively small magnitude. In large-scale drug screening, these transient, relatively small signal changes must be detected in multiple assays simultaneously.
Consequently, there is little margin for error in each single compound assay; an erroneous signal measurement by the detection system could result in elimination of a viable drug from further consideration. Additionally, signal measurement accuracy and sensitivity is essential in detecting small but significant differences in cellular responses to varying doses of compounds and in the response generated by an unknown compound as compared to a standard known drug.
Thus, there is a need for signal detection instrumentation that enables fully automated, high-volume assays of rapid, transient phenomena with sufficient sensitivity and the degree of accuracy required for applications such as drug screening.